Appearance Yellowish amorphous powder, lyophilized
Activity GradeⅢ 20U/mg-solid or more
(containing approx. 40% of stabilizers)
Contaminant NADPH oxidase ≤1.0×10⁻¹%
Stabilizers Sugars, FAD
Stability Stable at -20°C for at least One year (Fig.1)
Molecular weight 55,000~60,000
Michaelis constants 2.0×10⁻⁵M (p-Hydroxybenzoate), 4.0×10⁻⁵M (NADPH)
Structure One mol of FAD per mol of enzyme
Inhibitors Ag⁺, Hg⁺⁺, PCMB, SDS
Optimum pH 7.7-7.9(Fig.3)
Optimum temperature 35°C(Fig.4)
pH Stability pH 5.0-7.5 (25°C, 72hr)(Fig.5)
Thermal stability below 40°C (pH 6.0, 15min)(Fig.6)
Substrate specificity (Table 1)
Effect of various chemicals (Table 2)


This enzyme is useful for enzymatic determination of choline esterase when coupled with protocatechuate 3, 4-dioxygenase (PCO-302).



p-hydroxybenzoate hydroxylase

p-Hydroxybenzoate+NADPH+H⁺+O₂                                                  Protocatechuate+NADP⁺+H₂O

The disappearance of NADPH is measured at 340nm by spectrophotometry.

Unit definition:

One unit causes the oxidation of one micromole of NADPH per minute under the conditions described below.


A. Tris-malate buffer, pH 8.2 50mM [Dissolve 3.03g of Tris (M.W=121.14) in ca.300ml of H₂O and, after adjusting the pH to 8.2 at 25°C with 1.0M maleic acid, fill up to 500ml with H₂O.]
B. p-hydroxybenzoate solution 5.0mM [80mg p-hydroxybenzoate (Na salt)/100ml of buffer solution (A)] (Should be prepared fresh)
C. FAD solution 0.2mM [19mg FAD・Na₂/100ml or buffer solution (A)](Should be prepared fresh)
D. NADPH solution 3.0mM [272mg NADPH・Na₄・4H₂O/100ml of buffer solution (A)](Should be prepared fresh)
F. Enzyme diluent 50mM K-phosphate buffer, pH 6.0 containing 0.2% BSA


Concentration in assay mixture
Tris-malate buffer 49 mM
p-Hydroxybenzoate 0.49mM
FAD 20 µM

1. Prepare the following working solution (10 tests) in a brownish bottle and store on ice.

21.0 ml Buffer solution (A)
3.0 ml Substrate solution (B)
3.0 ml FAD solution (C)
3.0 ml NADPH solution (D)

2. Pipette 3.0ml of working solution into a cuvette (d=1.0cm) and equilibrate at 37°C for about 5 minutes.

3. Add 0.05ml of the enzyme solution* and mix by gentle inversion.

4. Record the decrease in optical density at 340nm against water for 3 to 4 minutes in a spectrophotometer thermostated at 37°C and calculate the ΔOD per minute from 1.5 to 3 minutes portion of the curve (ΔOD test). At the same time, measure the blank rate (ΔOD blank) by using the same method as the test except that the enzyme diluent (E) is added instead of enzyme solution.

* Dissolve the enzyme preparation in ice-cold enzyme diluent (E) (1.0mg/ml or more) and dilute to 0.2-0.6 U/ml with the same buffer, immediately before assay.


Activity can be calculated by using the following formula :

ΔOD/min (ΔOD test−ΔOD blank ) ×Vt × df

Volume activity (U/ml) =                                                               =ΔOD/min×9.8×df


Weight activity (U/mg)=(U/ml)×1/C

: Total volume (3.05ml)
: Sample volume (0.05ml)
: Millimolar extinction coefficient of NADPH (㎠/micromole)
: Light path length (cm)
: Dilution factor
: Enzyme concentration (c mg/ml)


  1. H.Shoun and K.Arima; Protein, Nucleic acid and Enzyme, 25, 820, (1980).
  2. K.Yano and K.Arima; Agric.Biol.Chem., 33, 689 (1969).
  3. K.Hosokawa and R.Y.Stanier; J.Biol.Chem., 241, 2453 (1966).
Table 1. Substrate Specificity of p-Hydroxybenzoate hydroxylase
Substrate (0.5mM) Relative activity(%) Substrate (0.5mM) Relative activity(%)
p-Hydroxybenzoic acid 100 Protocatechuic acid 3.3
Methyl-p-hydroxybenzoic acid <0.05 ß-Resorcylic acid 4.5
Ethyl-p-hydroxybenzoic acid <0.05
Gentisic acid <0.05
n-Propyl-p-hydroxybenzoic acid <0.05
p-Chlorobenzoic acid <0.05
m-Hydroxybenzoic acid <0.05 p-Aminobenzoic acid 0.12
o-Hydroxybenzoic acid <0.05    
Table 2. Effect of Various Chemicals on p-Hydroxybenzoate hydroxylase
(Residual activity after 1 hr-treatment at 30°C)
Chemical Concn.(mM) Residual
Chemical Concn.(mM) Residual
None 100 MIA 1.0 91
Metal salt 1.0   PCMB 1.0 3.7
CoCl₂   106
NaN 1.0 97
  94 NaF 1.0 96
  103 o-Phenanthroline 1.0 95
AgNO₃   0 α,α′-Dipyridyl 1.0 90
MgSO₄   107 EDTA 5.0 96
BaCl₂   107 Borate 50 104
FeCl₃   106 Tween 20 0.1% 91
MnCl₂   90 Brij 35 0.1% 101
NiCl₂   104 Span 20 0.1% 94
CaCl₂   97 Triton X-100 0.1% 97
SnCl₂   102
Na-cholate 0.1% 88
HgCl₂   1.1 SDS 0.05% 34
CrCl₂   93
CdCl₂   104      
FeSO₄   90      

MIA, Monoiodoacetate; PCMB, p-Chloromercuribenzoate; EDTA, Ethylenediaminetetraacetate; SDS, Sodium dodecyl sulfate.

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